P2U2 receptor antibodies

ABSTRACT

A novel subtype of the P 2 -purinergic receptor, referred to as the P 2U2  receptor, is disclosed. This receptor is activated by four of its agonists in the following order of specificity: UTP&gt;UDP&gt;ADP&gt;ATP. Nucleic acids encoding the receptor, antibodies that bind to the receptor and associated screening and therapeutic methods also are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation patent application under 37 C.F.R.§1.53(b), of application Ser. No. 09/410,738, filed Oct. 1, 1999 nowabandoned, which is a divisional of prior application Ser. No.08/749,707, filed on Nov. 15, 1996 (issued as U.S. Pat. No. 6,063,582),which is a continuation of prior application Ser. No. 08/559,524, filedon Nov. 15, 1995 (issued as U.S. Pat. No. 5,871,963) claiming priorityto provisional application no. 60/006,782, filed on Nov. 15, 1995, allof which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a new subtype of the P₂-purnergicreceptors, which is abundantly expressed in kidney and in many celllines of megakaryocytic or erythroleukemic origin. Referred to herein asthe P_(2U2) receptor, this receptor is activated by ATP, ADP, UTP andUDP. The P_(2U2) receptor can be used as a tool to screen for agonistsand antagonists that can either stimulate or block receptor activation.Such compounds have therapeutic utility in treating (1) diseases thatare caused by aberrant activation of this receptor, for example overstimulation or under stimulation of the receptor and (2) diseases whosesymptoms can be ameliorated by stimulating or inhibiting the activity ofthe P_(2U2) receptor.

The present invention also relates to the isolated entire human geneencoding the P_(2U2) receptor, methods for the recombinant production ofpurified P_(2U2) receptor proteins and the proteins made by thesemethods, antibodies against the whole P_(2U2) receptor or regionsthereof, vectors, nucleotide probes, and host cells transformed by genesencoding polypeptides having the P_(2U2) receptor activity, along withdiagnostic and therapeutic uses for these various reagents.

BACKGROUND OF THE INVENTION

Purinergic receptors are cell surface receptors that interact withextracellular adenine or uridine nucleotides and nucleosides. Thesereceptors are present throughout the central nervous system andperipheral tissues and play a role in numerous physiological responses.

The purinergic receptors are broadly divided into two major receptortypes, P₁ and P₂, which are defined by their level of interaction withthe adenine nucleotides and nucleosides. Where P₁ receptors areactivated by adenosine and exhibit a potency order ofadenosine>AMP>ADP>ATP, P₂ receptors are activated by ATP, UTP, ADP orUDP and exhibit a potency order of ATP≧ADP>AMP>adenosine. As more hasbecome known about the purinergic receptors and the wide range ofphysiological responses in which they play a role, the P₁- and P₂-typeclassifications were no longer sufficient to accurately portray thiscomplex family of receptors. Therefore, receptor subtype categories havebeen developed. For example, the P₂-type purinergic receptors are nowclassified as P_(2y)-, P_(2U)- , P_(2T)-, P_(2X)- and P_(2Z)-subtypes. Areview of the P₂-type purinergic receptors can be found in Harden, etal., Ann. Rev. Pharmacol. Toxicol. 35:541-579 (1995).

Classification of the P₂-type purinergic receptors has been difficultbecause there are no published selective P₂-receptor antagonists andthere are few ATP or ADP receptor-subtype specific agonists. Inaddition, it has been difficult to compare the relative order of potencyof P₂-purinergic receptor agonists. Hence, this subtype has presentednumerous challenges in the identification and characterization of itsmembers.

SUMMARY OF THE PRESENT INVENTION

One aspect of the invention is an isolated and purified polypeptidecomprising the amino acid sequence of FIG. 1 (SEQ ID NO:2).

Another aspect of the invention is an isolated and purified nucleic acidsequence encoding for the P_(2U2) receptor.

Yet another aspect of the invention is an isolated and purified nucleicacid sequence comprising the nucleotide sequence of FIG. 1 (SEQ IDNO:1).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the DNA (SEQ ID NO:1) and deduced amino acid sequence (SEQ IDNO:2) of the human P_(2U2) receptor.

FIG. 2 is a comparison of the amino acid sequence (SEQ ID NO:2) of thehuman P_(2U2) receptor with the amino acid sequence of the human P_(2U)receptor (Parr, et al., Proc. Natl. Acad. Sci. USA 91:3275-3279 (1994))(SEQ ID NO:3) and the bovine P₂Y₁ receptor (Henderson, et al. BBRC212:648-656 (1995) (SEQ ID NO:4). The Parr P_(2U) receptor is referredto in FIG. 2 as “P_(2U1)”.

FIG. 3 shows representative chloride currents obtained from oocytesinjected with cRNA for the receptor and challenged with a variety ofpurinergic agonists (ADP, ATP, UTP, UDP).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods and materials useful in theregulation of the renal system in mammals. Recent studies provideevidence that extracellular nucleotides influence the renalmicrovasculature. See Inscho, et al., FASEB Journal 8:319-328 (1994).The isolation, recombinant production and characterization of thepurinergic receptor of the invention allows for the effective regulationof these functions.

Before proceeding further with a description of the specific embodimentsof the present invention, a number of terms will be defined.

The terms “substantially pure” and “isolated” are used herein todescribe a protein that has been separated from the native contaminantsor components that naturally accompany it. Typically, a monomericprotein is substantially pure when at least about 60 to 70% of a sampleexhibits a single polypeptide backbone. Minor variants or chemicalmodifications typically share approximately the same polypeptidesequence. A substantially pure protein will typically comprise overabout 85 to 90% of a protein sample, preferably will comprise at leastabout 95%, and more preferably will be over about 99% pure. Purity istypically measured on a polyacrylamide gel, with homogeneity determinedby staining. For certain purposes, high resolution will be desired andHPLC or a similar means for purification utilized. However, for mostpurposes, a simple chromatography column or polyacrylamide gel will beused to determine purity. Whether soluble or membrane bound, the presentinvention provides for substantially pure preparations. Various methodsfor their isolation from biological material may be devised, based inpart upon the structural and functional descriptions contained herein.In addition, a protein that is chemically synthesized or synthesized ina cellular system that is different from the cell from which itnaturally originates, will be substantially pure. The term is also usedto describe receptors and nucleic acids that have been synthesized inheterologous mammalian cells or plant cells, E. coli and otherprokaryotes.

As used herein, the terms “hybridization” (hybridizing) and“specificity” (specific for) in the context of nucleotide sequences areused interchangeably. The ability of two nucleotide sequences tohybridize to each other is based upon a degree of complementarity of thetwo nucleotide sequences, which in turn is based on the fraction ofmatched complementary nucleotide pairs. The more nucleotides in a givensequence that are complementary to another sequence, the greater thedegree of hybridization of one to the other. The degree of hybridizationalso depends on the conditions of stringency which include temperature,solvent ratios, salt concentrations, and the like. In particular,“selective hybridization” pertains to conditions in which the degree ofhybridization of a polynucleotide of the invention to its target wouldrequire complete or nearly complete complementarity. The complementaritymust be sufficiently high so as to assure that the polynucleotide of theinvention will bind specifically to the target relative to binding othernucleic acids present in the hybridization medium. With selectivehybridization, complementarity will be 90-100%, preferably 95-100%, morepreferably 100%.

The present invention relates to a new purnergic receptor of the P₂subclass, which is referred to herein as the P_(2U2) receptor. FIG. 1shows the DNA sequence of the clone encoding the P_(2U2) receptor alongwith the deduced amino acid sequence. The amino acid sequence shown inFIG. 1 includes four putative extracellular domains (the NH₂-terminusand ECD I-ECD III) and seven putative transmembrane regions (TM I-TMVII). As used herein, the “P_(2U2) receptor” refers to receptor in anyanimal species sharing a common biological activity with the humanreceptor contained in the clone described in Example 1 herein. This“common biological activity” includes but is not limited to an effectoror receptor function or cross-reactive antigenicity. Using the nativeDNA encoding the human form of this receptor, the P_(2U2) receptors inother species, may be obtained.

Because the P_(2U2) receptor is activated by UTP, it is classified as aP₂-type purinergic receptor. Hydrophobicity/hydrophilicity plots of theP_(2U2) receptor sequence shown in FIG. 1 suggest that the P_(2U2)receptor has 7 putative transmembrane domains. This, along with thefollowing characteristics, are consistent with characteristics that areobserved in other P₂-type purinergic receptors:

seven putative α-helical transmembrane-spanning structures;

amino terminus located on the extracellular side of the membrane;

carboxy terminus located on the intracellular side of the membrane; and

conservation of sequence in the transmembrane spanning domains ascompared with other P₂-purinergic receptors.

It has been found that the P_(2U2) receptor is expressed in many celllines of megakaryocytic or erythroleukemic origin. In addition, theP_(2U2) receptor is expressed, at the RNA level, predominantly in thekidney. This receptor is unusual in that, although most purinergicreceptors are present in the brain, the P_(2U2) receptor has not beenfound to be expressed in human brain tissue. The tissue distribution ofthe P_(2U2) receptor is described in Example 3.

Some P₂ receptors have a strong preference for one nucleotide.Alternately, they may be activated by several nucleotides but thespecificity for one nucleotide is usually an order of magnitude greaterthan for the other nucleotides. The P_(2U2) receptor is activated byATP, ADP, UTP and UDP when expressed in Xenopus oocytes, with thefollowing order of specificity:

UTP>UDP>ADP>ATP

However, unlike for other P₂ receptors, the potency of ATP, ADP, UTP andUDP as agonists for the P_(2U2) receptor are close in value, with a merefive-fold difference:

One aspect of the present invention also relates to the human geneencoding the P_(2U2) receptor, which has both diagnostic and therapeuticuses as are described below. Included within this invention are proteinsor peptides having substantial homology with the amino acid sequence ofFIG. 1.

Ordinarily, the P_(2U2) receptors and analogs thereof claimed hereinwill have an amino acid sequence having at least 75% amino acid sequenceidentity with the P_(2U2) receptor sequence disclosed in FIG. 1, morepreferably at least 80%, even more preferably at least 90%, and mostpreferably at least 95%. Identity or homology with a sequence is definedherein as the percentage of amino acid residues in the candidatesequence that are identical with the sequence of the P_(2U2) receptor,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent homology, and not considering anyconservative substitutions as part of the sequence identity. None ofN-terminal, C-terminal or internal extensions, deletions, or insertionsof the P_(2U2) receptor sequence shall be construed as affectinghomology.

Thus, the claimed P_(2U2) receptor and analog molecules that are thesubject of this invention include molecules having the P_(2U2) receptoramino acid sequence; fragments thereof having a consecutive sequence ofat least 10, 15, 20, 25, 30 or 40 amino acid residues from the P_(2U2)receptor sequence of FIG. 1; amino acid sequence variants of the P_(2U2)receptor sequence of FIG. 1 wherein an amino acid residue has beeninserted N- or C-terminal to, or within, (including parallel deletions)the P_(2U2) receptor sequence or its fragments as defined above; aminoacid sequence variants of the P_(2U2) receptor sequence of FIG. 1 or itsfragments as defined above which have been substituted by at least oneresidue.

P_(2U2) receptor polypeptides include those containing predeterminedmutations by, e.g., homologous recombination, site-directed or PCRmutagenesis, and P_(2U2) receptor polypeptides of other animal species,including but not limited to rabbit, rat, murine, porcine, bovine,ovine, equine and non-human primate species, and alleles or othernaturally occurring variants of the P_(2U2) receptor of the foregoingspecies and of human sequences; derivatives of the commonly knownP_(2U2) receptor or its fragments wherein the P_(2U2) receptor or itsfragments have been covalently modified by substitution, chemical,enzymatic, or other appropriate means with a moiety other than anaturally occurring amino acid (for example a detectable moiety such asan enzyme or radioisotope); glycsylation variants of the P_(2U2)receptor (insertion of a glycosylation site or deletion of anyglycosylation site by deletion, insertion or substitution of appropriateamino acid); and soluble forms of the P_(2U2) receptor. This inventionalso includes tagging the P_(2U2) receptor, in particular for use inpurification or diagnostic application. Types and methods of tagging arewell known in the art, for example, the use of hexa-histidine tags.

Most sequence modifications, including deletions and insertions, andsubstitutions in particular, are not expected to produce radical changesin the characteristics of the P_(2U2) receptor. However, when it isdifficult to predict the exact effect of the sequence modification inadvance of making the change, one skilled in the art will appreciatethat the affect of any sequence modification will be evaluated byroutine screening assays.

P_(2U2) receptor peptides may be purified using techniques of classicalprotein chemistry, such as are well known in the art. For example, alectin affinity chromatography step may be used, followed by a highlyspecific ligand affinity chromatography procedure that utilizes a ligandconjugated to biotin through the cysteine residues of the ligand.Alternately, a hexa-histidine tagged receptor may be purified usingnickel column chromatography.

The nomenclature used to describe the peptide compounds of the inventionfollows the conventional practice where the N-terminal amino group isassumed to be to the left and the carboxy group to the right of eachamino acid residue in the peptide. In the formulas representing selectedspecific embodiments of the present invention, the amino- andcarboxy-terminal groups, although often not specifically shown, will beunderstood to be in the form they would assume at physiological pHvalues, unless otherwise specified. Thus, the N-terminal H⁺ ₂ andC-terminal O− at physiological pH are understood to be present thoughnot necessarily specified and shown, either in specific examples or ingeneric formulas. Free functional groups on the side chains of the aminoacid residues can also be modified by amidation, acylation or othersubstitution, which can, for example, change the solubility of thecompounds without affecting their activity.

In the peptides shown, each gene-encoded residue, where appropriate, isrepresented by a single letter designation, corresponding to the trivialname of the amino acid, in accordance with the following conventionallist:

One-Letter Amino Acid Symbol Three-letter Alanine A Ala Arginine R ArgAsparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine Q GlnGlutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val

The amino acids not encoded genetically are abbreviated as indicated inthe discussion below.

In the specific peptides shown in the present application, the L-form ofany amino acid residue having an optical isomer is intended unless theD-form is expressly indicated by a dagger superscript (†). Thisinvention also contemplates non-naturally occurring amino acids(typically those which are not naturally encoded) as are well known inthe art.

The compounds of the invention are peptides which are partially definedin terms of amino acid residues of designated classes. Amino acidresidues can be generally subclassified into four major subclasses asfollows:

Acidic: The residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous medium at physiologicalpH.

Basic: The residue has a positive charge due to association with H ionat physiological pH and the residue is attracted by aqueous solution soas to seek the surface positions in the conformation of a peptide inwhich it is contained when the peptide is in aqueous medium atphysiological pH.

Neutral/nonpolar: The residues are not charged at physiological pH andthe residue is repelled by aqueous solution so as to seek the innerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium. These residues are also designated“hydrophobic” herein.

Neutral/polar: The residues are not charged at physiological pH, but theresidue is attracted by aqueous solution so as to seek the outerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium.

It is understood, of course, that in a statistical collection ofindividual residue molecules some molecules will be charged, and somenot, and there will be an attraction for or repulsion from an aqueousmedium to a greater or lesser extent. To fit the definition of“charged,” a significant percentage (at least approximately 25%) of theindividual molecules are charged at physiological pH. The degree ofattraction or repulsion required for classification as polar or nonpolaris arbitrary and, therefore, amino acids specifically contemplated bythe invention have been classified as one or the other. Most amino acidsnot specifically named can be classified on the basis of known behavior.

Amino acid residues can be further subclassified as cyclic or noncyclic,and aromatic or nonaromatic, self-explanatory classifications withrespect to the side chain substituent groups of the residues, and assmall or large. The residue is considered small if it contains a totalof 4 carbon atoms or less, inclusive of the carboxyl carbon. Smallresidues are, of course, always nonaromatic.

For the naturally occurring protein amino acids, subclassificationaccording to the foregoing scheme is as follows:

Acidic: Aspartic acid and Glutamic acid

Basic/noncyclic: Arginine and Lysine

Basic/cyclic: Histidine

Neutra/polar/small: Glycine, serine and cysteine

Neutral/nonpolar/small: Alanine

Neutral/polar/large/nonaromatic: Threonine, Asparagine and Glutamine

Neutral/polar/large aromatic: Tyrosine

Neutral/nonpolar/large/nonaromatic: Valine, Isoleucine, Leucine andMethionine

Neutral/nonpolar/large/aromatic: Phenylalanine, and Tryptophan

The gene-encoded secondary amino acid proline, although technicallywithin the group neutral/nonpolar/large/cyclic and nonaromatic, is aspecial case due to its known effects on the secondary conformation ofpeptide chains, and is not, therefore, included in this defined group.

Certain commonly encountered amino acids, which are not encoded by thegenetic code, include, for example, beta-alanine (beta-Ala), or otheromega-amino acids, such as 3-amino propionic, 2,3-diamino propionic(2,3-diaP), 4-amino butyric and so forth, alpha-aminisobutyric acid(Aib), sarcosine (Sar), omithine (Om), citrulline (Cit), t-butylalanine(t-BuA), t-butylglycine (t-BuG), N-methylisoleucine (N-Melle),phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle),cysteic acid (Cya) 2-naphthylalanine (2-Nal);1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);β-2-thienylalanine (Thi); and methionine sulfoxide (MSO). These alsofall conveniently into particular categories.

Based on the above definitions,

Sar, beta-Ala, 2,3-diaP and Aib are neutral/nonpolar/small;

t-BuA, t-BuG, N-Melle, Nle, Mvl and Cha areneutral/nonpolar/large/nonaromatic;

Om is basic/noncyclic;

Cya is acidic;

Cit, Acetyl Lys, and MSO are neutral/polar/large/nonaromatic; and

Phg, Nal, Thi and Tic are neutral/nonpolar/large/aromatic.

The various omega-amino acids are classified according to size asneutral/nonpolar/small (beta-Ala, i.e., 3-aminopropionic,4-aminobutyric) or large (all others).

Other amino acid substitutions of those encoded in the gene can also beincluded in peptide compounds within the scope of the invention and canbe classified within this general scheme according to their structure.

All of the compounds of the invention, when an amino acid forms theC-terminus, may be in the form of the pharmaceutically acceptable saltsor esters. Salts may be, for example, Na⁺, K⁺, Ca⁺², Mg⁺² and the like;the esters are generally those of alcohols of 1-6C.

In all of the peptides of the invention, one or more amide linkages(—CO—NH—) may optionally be replaced with another linkage which is anisostere such as —CH₂NH—, —CH₂S—, —CH₂CH₂, —CH═CH—(cis and trans),—COCH₂—, —CH(OH)CH₂— and —CH₂SO—. This replacement can be made bymethods known in the art. The following references describe preparationof peptide analogs which include these alternative-linking moieties:Spatola, Vega Data 1(3) “Peptide Backbone Modifications” (generalreview) (March 1983); Spatola, in “Chemistry and Biochemistry of AminoAcids Peptides and Proteins,” B. Weinstein, eds., Marcel Dekker, NewYork, p. 267 (1983) (general review); Morley, J. S., Trends Pharm Sci,pp. 463-468 (general review) (1980); Hudson, et al., Int J Pept Prot Res14:177-185 (—CH₂NH—, —CH₂CH₂—) (1979); Spatola, et al., Life Sci38:1243-1249 (—CH ₂—S) (1986); Hann, J Chem Soc Perkin Trans I 307-314(—CH—CH—, cis and trans) (1982); Alrnquist, et al., J Med Chem23:1392-1398 (—COCH₂—) (1980); Jennings-White, et al., Tetrahedron Lett23:2533 (—COCH₂—) (1982); Szelke, et al., European Application EP 45665(1982) CA:97:39405 (1982) (—CH(OH)CH₂—); Holladay, et al., TetrahedronLett 4:4401-4404 (—C(OH)CH₂—) (1983); and Hruby, Life Sci 31:189-199(—CH₂—S—) (1982).

The invention provides methods and materials useful in assay systems todetermine the ability of candidate pharmaceuticals to affect theactivity of the P_(2U2) receptor. The isolation, recombinant productionand characterization of the P_(2U2) receptor allows for the design ofassay systems using the P_(2U2) receptor as a substrate and usingagonists and antagonists for the receptor as control reagents in theassay.

One embodiment of the invention relates to recombinant materialsassociated with the production of the P_(2U2) receptor. These includetransfected cells that can be cultured so as to display or express theP_(2U2) receptor on its surface, thus providing an assay system for theinteraction of materials with the native P_(2U2) receptor where thesecells or relevant fragments of the P_(2U2) receptor are used as ascreening tool to evaluate the effect of various candidate compounds onthe P_(2U2) receptor activity in vivo, as is described below. Suitablecells include Xenopus oocytes and most mammalian cell lines.

Recombinant production of the P_(2U2) receptor involves using a nucleicacid sequence that encodes the P_(2U2) receptor, as is set forth in FIG.1, or its degenerate analogs. The nucleic acid can be prepared either byretrieving the native sequence, as described below, or by usingsubstantial portions of the known native sequence as a probe, or it canbe synthesized de novo using procedures that are well known in the art.

The nucleic acid may be ligated into expression vectors suitable for thedesired host and then transformed into compatible cells. Alternatively,nucleic acids may be introduced directly into a host cell by techniquessuch as are well known in the art. The cells are cultured underconditions favorable for the expression of the gene encoding the P_(2U2)receptor and cells displaying the receptor on the surface are thenharvested. Suitable cells include E. coli, Chinese Hamster Ovary cells,human Jurkat T-cell line, the rat-2 fibroblast cell line, humanastocytoma 1321N1 cell line and insect cell lines such as Sf-9.

This invention also relates to nucleic acids that encode or arecomplementary to a P_(2U2) receptor polypeptide. These nucleic acids canthen be used to produce the polypeptide in recombinant cell culture fordiagnostic use or for potential therapeutic use. In still other aspects,the invention provides an isolated nucleic acid molecule encoding aP_(2U2) receptor, either labeled or unlabeled, or a nucleic acidsequence that is complementary to, or hybridizes under stringentconditions to, a nucleic acid sequence encoding a P_(2U2) receptor. Theisolated nucleic acid molecule of the invention excludes nucleic acidsequences which encode, or are complementary to nucleic acid sequencesencoding, other known purinergic receptors which are not P_(2U2)receptors, such as the human P_(2U), and the chicken and bovine P_(2Y1)receptors, and the like.

This invention also provides a replicable vector comprising a nucleicacid molecule encoding a P_(2U2) receptor operably linked to controlsequences recognized by a host transformed by the vector; host cellstransformed with the vector; and a method of using a nucleic acidmolecule encoding a P_(2U2) receptor to effect the production of aP_(2U2) receptor on the cell surface, comprising expressing the nucleicacid molecule in a culture of the transformed host cells and recoveredfrom the cells. The nucleic acid sequence is also useful inhybridization assays for P_(2U2) receptor-encoding nucleic acidmolecules.

In still further embodiments of the invention, a method is described forproducing P_(2U2) receptors comprising inserting into the DNA of a cellcontaining the nucleic acid sequence encoding a P_(2U2) receptor atranscription modulatory element (such as an enhancer or a silencer) insufficient proximity and orientation to the P_(2U2) receptor codingsequence to influence transcription thereof, with an optional furtherstep comprising culturing the cell containing the transcriptionmodulatory element and the P_(2U2) receptor-encoding nucleic acidsequence.

This invention also covers a cell comprising a nucleic acid sequenceencoding a P_(2U2) receptor and an exogenous transcription modulatoryelement in sufficient proximity and orientation to the above codingsequence to influence transcription thereof and a host cell containingthe nucleic acid sequence encoding a P_(2U2) receptor operably linked toexogenous control sequences recognized by the host cell.

This invention provides a method for obtaining cells having increased ordecreased transcription of the nucleic acid molecule encoding a P_(2U2)receptor, comprising: providing cells containing the nucleic acidmolecule; introducing into the cells a transcription modulating element;and screening the cells for a cell in which the transcription of thenucleic acid molecule is increased or decreased.

P_(2U2) receptor nucleic acids for use in the invention can be producedas follows. A P_(2U2) receptor “nucleic acid” is defined as RNA or DNAthat encodes a P_(2U2) receptor, or is complementary to nucleic acidsequence encoding a P_(2U2) receptor, or hybridizes to such nucleic acidand remains stably bound to it under stringent conditions, or encodes apolypeptide sharing at least 75% sequence identity, preferably at least80%, and more preferably at least 85%, with the translated amino acidsequence shown in FIG. 1. It is typically at least about 10 nucleotidesin length and preferably has P_(2U2) receptor related biological orimmunological activity. Specifically contemplated are genomic DNA, cDNA,mRNA and antisense molecules, as well as nucleic acids based onalternative backbone or including alternative bases whether derived fromnatural sources or synthesized.

“Stringent conditions” are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0,015M NaCl/0.0015M sodiumtitrate/0.1% NaDodSO₄ at 50° C., or (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C. Another example is use of 50% formamide, 5×SSC (0.75MNaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA(50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at42° C. in 0.2×SSC and 0.1% SDS.

“Isolated” nucleic acid will be nucleic acid that is identified andseparated from contaminant nucleic acid encoding other polypeptides fromthe source of nucleic acid. The nucleic acid may be labeled fordiagnostic and probe purposes, using any label known and described inthe art as useful in connection with diagnostic assays.

Of particular interest is a P_(2U2) receptor nucleic acid that encodes afull-length molecule, including but not necessarily the native signalsequence thereof. Nucleic acid encoding full-length protein is obtainedby screening selected cDNA or genomic libraries using the deduced aminoacid sequence disclosed herein for the first time, and, if necessary,using conventional primer extension procedures to secure DNA that iscomplete at its 5′ coding end. Such a clone is readily identified by thepresence of a start codon in reading frame with the original sequence.

DNA encoding an amino acid sequence variant of a P_(2U2) receptor isprepared as described below or by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of a P_(2U2) receptor.

Techniques for isolating and manipulating nucleic acids are disclosedfor example by the following documents: U.S. Pat. Nos. 5,030,576,5,030,576 and International Patent Publications WO94/11504 andWO93/03162. See, also, Sambrook, et al., “Molecular Cloning: ALaboratory Manual”, 2nd Edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 1989, and Ausubel, et al. “Current Protocols in MolecularBiology”, Vol. 2, Wiley-Interscience, New York, 1987.

As mentioned above, the availability of the isolated cells providing theP_(2U2) receptor on their surface and the availability of therecombinant DNA encoding the P_(2U2) receptor which permits display andexpression of the receptor on host cell surfaces, all makes such cellsavailable as a valuable tool for evaluating the ability of candidateagonists or antagonists to bind to the receptor and thus contribute tothe receptor's activation or deactivation. In this manner, the inventionis related to assay systems which utilize an isolated or a recombinantlyproduced P_(2U2) receptor to screen for agonist and antagonist activityof candidate drugs. This assay is especially useful in assuring thatthese candidate therapeutic agents have the desired effect of eitheractivating or inhibiting the P_(2U2) receptor. Determination of theseproperties is essential in evaluating the specificity of drugs intendedfor binding other related receptors.

The host cells are typically animal cells, most typically mammaliancells. In order to be useful in the assays, the cells must haveintracellular mechanisms which permit the receptor to be displayed onthe cell surface. Particularly useful cells for use in the method of theinvention are Xenopus laevis frog oocytes, which typically utilize cRNArather than standard recombinant expression systems proceeding from theDNA encoding the desired protein. Capped RNA (at the 5′ end) istypically produced from linearized vectors containing DNA sequencesencoding the receptor. The reaction is conducted using RNA polymeraseand standard reagents. cRNA is recovered, typically usingphenol/chloroform precipitation with ethanol and injected into theoocytes.

The animal host cells expressing the DNA encoding the P_(2U2) receptoror the cRNA-injected oocytes are then cultured to effect the expressionof the encoding nucleic acids so as to produce the P_(2U2) receptordisplay on the cell surface. These cells then are used directly inassays for assessment of a candidate drug to bind, antagonize, oractivate the receptor.

One method of evaluating candidates as potential therapeutic agentstypically involves a binding assay in which the candidate (such as apeptide or a small organic molecule) would be tested to measure if, orto what extent, it binds the P_(2U2) receptor. Preferably, a mammalianor insect cell line is used to express the P_(2U2) receptor or plasmamembrane preparations thereof, will be used in a binding assay. Forexample, a candidate antagonist competes for binding to the P_(2U2)receptor with either a labeled nucleotide agonist or antagonist. Varyingconcentrations of the candidate are supplied, along with a constantconcentration of the labeled agonist or antagonist. The inhibition ofbinding of the labeled material can then be measured using establishedtechniques. This measure rent is then correlated to determine the amountand potency of the candidate that is bound to the P_(2U2) receptor.

Another method of evaluating candidates for potential therapeuticapplications typically involves a functional assay in which thecandidate's effect upon cells expressing the recombinant P_(2U2)receptor is measured, rather than simply determining its ability to bindthe P_(2U2) receptor. Suitable functional assays include those thatmeasure calcium mobilization (⁴⁵Ca efflux or measurements ofintracellular Ca⁺² concentration with fluorescent dyes such as fura-2)and voltage clamp, described below.

For example, agonist-induced increases in ⁴⁵Ca release by oocytesexpressing cRNA encoding the P_(2U2) receptor or other mammalianrecombinant cells producing the P_(2U2) receptor can be measured by thetechniques described by Williams, et al., Proc Natl Acad Sci USA85:4939-4943 (1988). Intracellular calcium pools are labeled byincubating groups of 30 oocytes in 300 μl calcium-free modified Barth'ssolution (MBSH) containing 50 μCi ⁴⁵CaCl₂ (10-40 mCi/mg Ca; Amersham)for 4 hours at room temperature. The labeled oocytes or cells arewashed, then incubated in MBSH II without antibiotics for 90 minutes.Groups of 5 oocytes are selected and placed in individual wells in a24-well tissue culture plate containing 0.5 m/well MBSH II withoutantibiotics. This medium is removed and replaced with fresh medium every10 minutes; the harvested medium is analyzed by scintillation countingto determine ⁴⁵Ca released by the oocytes during each 10-minuteincubation. The 10-minute incubations are continued until a stablebaseline of ⁴⁵Ca release per unit time is achieved. Two additional10-minute collections are obtained, then test medium including agonistis added and ⁴⁵Ca release determined.

Using the above assay, the ability of a candidate drug to activate theP_(2U2) receptor can be tested directly. In this case, the agonists ofthe invention are used as controls. In addition, by using the agonistsof the invention to activate the recombinant receptor, the effect of thecandidate drug on this activation can be tested directly. Cellsexpressing the nucleic acids encoding the receptor are incubated in theassay in the presence of agonist with and without the candidatecompound. A diminution in activation in the presence of the candidatewill indicate an antagonist effect. Conversely, the ability of acandidate drug to reverse the antagonist effects of an antagonist of theinvention may also be tested.

As indicated above, receptor activation can also be measured by means ofthe two-electrode voltage clamp assay. In this assay, agonist-inducedinward chloride currents are measured in voltage-clamped oocytes thatexpress the P_(2U2) receptor. The technique suitable for use in theinstant invention is described by Julius, et al, Science 241:558-563(1988).

The P_(2U2) receptor also has utility in assays for the diagnosis ofrenal system diseases and disorders by detection, in tissue samples, ofaberrant expression of the P_(2U2) receptor.

Another aspect of the invention relates to P_(2U2) receptor agoniststhat imitate the activated form of the P_(2U2) receptor. These agonistsare useful as control reagents in the above-mentioned assays to verifythe workability of the assay system. In addition, agonists for theP_(2U2) receptor may exhibit useful effects in vivo in treating kidneydisease.

Another aspect of the invention relates to P_(2U2) receptor antagoniststhat are modified forms of P_(2U2) receptor peptides. Such antagonistsbind to the P_(2U2) receptor, but do not activate it, and preventreceptor activation by naturally occurring ligands by blocking theirbinding to the receptor. Another group of compounds within the scope ofthe invention, are antagonists of the P_(2U2) receptor ligands, i.e.,these are ligand inhibitors. Both these types of antagonists findutility in diminishing or mediating ligand-mediated events such ascalcium release. Yet another second group of antagonists includesantibodies designed to bind specific portions of the P_(2U2) receptorprotein. In general, these are monoclonal antibody preparations whichare highly specific for any desired region of the P_(2U2) receptor. Theantibodies, which are explained in greater detail below, are also usefulin immunoassays for the receptor protein, for example, in assessingsuccessful expression of the gene in recombinant systems.

In both the agonists and antagonists, a preferred embodiment is thatclass of compounds having amino acid sequences that are encoded by theP_(2U2) receptor gene. Preferably, the agonists and antagonists haveamino acid sequences, in whole or in part, corresponding to theextracellular domains of the P_(2U2) receptor. For example, preferredpeptides of the invention correspond, in whole or in part, to either theamino terminus, which is amino acid no 1, methionine (M) to amino acidno 23, lysine (K) (SEQ ID NO:5); ECD I, which is amino acid no 83,tyrosine (Y) to amino acid no 99, arginine (R) (SEQ ID NO:6); ECD II,which is amino acid no 162, asparagine (N) to amino acid no 183,tyrosine(Y) (SEQ ID NO:7); or ECD III, which is amino acid no 257,alanine (A) to amino acid no 276, phenylalanine (F) (SEQ ID NO:8). Alsoincluded in the invention are isolated DNA molecules that encode thesespecific peptides. Accordingly, the invention pertains to isolated DNAmolecules encoding human P_(2U2) receptor peptides comprising the aminoacid sequence of FIG. 1 from amino acid no 1, methionine to amino acidno 23, lysine (SEQ ID NO:5); from amino acid no 83, tyrosine to aminoacid no 99, arginine (SEQ ID NO:6); from amino acid no 162, asparagineto amino acid no 183, tyrosine (SEQ ID NO:7); and from amino acid no257, alanine to amino acid no 276, phenylalanine (SEQ ID NO:8).

The invention also includes agonists and antagonists that affectreceptor function by binding to one of the intracellular (ICD) domainsof the receptor. For example, preferred peptides within this aspect ofthe invention would correspond, in whole or in part, to either ICD I,which is amino acid no 50, phenylalanine (F) to amino acid no 60,isoleucine (I) (SEQ ID NO:11); ICD II, which is amino acid no 120,arginine (R) to amino acid no 141, leucine (L) (SEQ ID NO:12); ICD III,which is amino acid no 208, tyrosine (Y) to amino acid no 233, leucine(L) (SEQ ID NO:13); or to the carboxy terminus, which is amino acid no301, histidine (H) to amino acid no 334, lysine (K) (SEQ ID NO:14). Alsoincluded in the invention are isolated DNA molecules that encode thesespecific peptides. Accordingly, the invention pertains to isolated DNAmolecules encoding human P_(2U2) receptor peptides comprising the aminoacid sequence of FIG. 1 from amino acid no 50, phenylalanine to aminoacid no 60, isoleucine (SEQ ID NO:11); amino acid no 120, arginine toamino acid no 141, leucine (SEQ ID NO:12); amino acid no 208, tyrosineto amino acid no 233, leucine (SEQ ID NO:13); and amino acid no 301,histidine (H) to amino acid no 334, lysine (K) (SEQ ID NO:14).

Also included are those compounds where one, two, three or more of theamino acid residues are replaced by one which is not encodedgenetically. In other purinergic receptors, the third, sixth and seventhtransmembrane (“TM”) regions have been shown to play a role in ligandbinding. See Erb, et al. JBC 270:4185-4188 (1995). Accordingly, it isexpected that the amino acid sequences of the TM III, TM VI and TM VIIregions of the P_(2U2) receptor, in whole or in part, will beparticularly useful in designed antibodies or peptides that can bind thereceptor and block ligand binding.

The peptide agonists and antagonists of the invention are preferablyabout 10-100 amino acids in length, more preferably 25-75 amino acids inlength. These peptides can be readily prepared using standard solidphase or solution phase peptide synthesis, as is well known in the art.In addition, the DNA encoding these peptides can be synthesized usingcommercially available oligonucleotide synthesis instrumentation andrecombinantly produced using standard recombinant production systems.Production using solid phase peptide synthesis is required when non-geneencoded amino acids are to be included in the peptide.

Another aspect of the invention pertains to antibodies, which have bothdiagnostic and therapeutic uses. Antibodies are able to act asantagonists or agonists by binding specific regions of the P_(2U2)receptor. The antibodies can be monoclonal or polyclonal, but arepreferably monoclonal antibodies that are highly specific for thereceptor and can be raised against the whole P_(2U2) receptor or regionsthereof. Preferably, the antibodies are obtained by immunization ofsuitable mammalian subjects (typically rabbit, rat, mouse, goat, human,etc.) with peptides containing as antigenic regions those portions ofthe P_(2U2) receptor intended to be targeted by the antibodies. Criticalregions include any region(s) of proteolytic cleavage, any segment(s) ofthe extracellular segment critical for activation, and the portions ofthe sequence which form the extracellular loops. These antibodies alsofind utility in immunoassays that measure the presence of the P_(2U2)receptor, for example in immunoassays that measure gene expression.

The antibodies of the present invention can be prepared by techniquesthat are well known in the art. Antibodies are prepared by immunizingsuitable mammalian hosts in appropriate immunization protocols using thepeptide haptens (immunogen) alone, if they are of sufficient length, or,if desired, or if required to enhance immunogenicity, conjugated tosuitable carriers. The immunogen will typically contain a portion of theP_(2U2) receptor that is intended to be targeted by the antibodies.Critical regions include those regions corresponding to theextracellular domains of the P_(2U2) receptor protein. Methods forpreparing immunogenic conjugates with carriers such as bovine serumalbumin, keyhole limpet hemocyanin, or other carrier proteins are wellknown in the art. In some circumstances, direct conjugation using, forexample, carbodiimide reagents may be effective; in other instanceslinking reagents such as those supplied by Pierce Chemical Co.,Rockford, Ill., may be desirable to provide accessibility to the hapten.The hapten can be extended at the amino or carboxy terminus with acysteine residue or interspersed with cysteine residues, for example, tofacilitate linking to carrier. The desired immunogen is administered toa host by injection over a suitable period of time using suitableadjuvants followed by collection of sera. Over the course of theimmunization schedule, titers of antibodies are taken to determine theadequacy of antibody formation.

Polyclonal antibodies are suitable for many diagnostic and researchpurposes and are easily prepared. Monoclonal antibodies are oftenpreferred for therapeutic applications and are prepared by continuoushybrid cell lines and collection of the secreted protein. Immortalizedcell lines that secrete the desired monoclonal antibodies can beprepared by the method described in Kohler and Milstein, Nature256:495-497 (1975) or modifications which effect immortalization oflymphocytes or spleen cells, as is generally known. The immortalizedcell lines are then screened by immunoassay techniques in which theantigen is the immunogen or a cell expressing the P_(2U2) receptor onits surface. Cells that are found to secrete the desired antibody, canthen be cultured in vitro or by production in the ascites fluid. Theantibodies are then recovered from the culture supernatant or from theascites supernatant.

Alternately, antibodies can be prepared by recombinant means, i.e., thecloning and expression of nucleotide sequences or mutagenized versionsthereof that at a minimum code for the amino acid sequences required forspecific binding of natural antibodies. Antibody regions that bindspecifically to the desired regions of receptor can also be produced aschimeras with regions of multiple species origin.

Antibodies may include a complete immunoglobulin or a fragment thereof,and includes the various classes and isotypes such as IgA, IgD, IgE,IgG1, IgG2a, IgG2b, IgG3 and IgM. Fragments include Fab, Fv, F(ab′)₂,Fab′, and so forth. Fragments of the monoclonals or the polyclonalantisera which contain the immunologically significant portion can beused as antagonists, as well as the intact antibodies. Use ofimmunologically reactive fragments, such as the Fab, Fab′, or F(ab′)₂fragments is often preferable, especially in a therapeutic context, asthese fragments have different immunogenicity than the wholeimmunoglobulin, and do not carry the biological activity of animmunoglobulin constant domain.

The antibodies thus produced are useful not only as potential agonist orantagonists for the receptor, filling the role of agonist or antagonistin the assays of the invention, but are also useful in immunoassays fordetecting the activated receptor. As such these antibodies can becoupled to imaging agents for administration to a subject to allowdetection of localized antibody to ascertain the position of P_(2U2)receptors in either activated or unactivated form. In addition, thesereagents are useful in vitro to detect, for example, the successfulproduction of the P_(2U2) receptor deployed at the surface of therecombinant host cells.

Yet another aspect of the invention relates to pharmaceuticalcompositions containing the compounds of the invention. The agonists andantagonists of the invention have therapeutic utility in (1) treatingdiseases caused by aberrant activation of this receptor in tissues whereit is customarily found, for example in the kidney and (2) treatingdiseases whose symptoms can be ameliorated by stimulating or inhibitingthe activity of the P_(2U2) receptor.

The peptide agonists and antagonists of the invention can beadministered in conventional formulations for systemic administrationsuch as is well known in the art. Typical formulations may be found, forexample, in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton Pa., latest edition.

Preferred forms of systemic administration include injection, typicallyby intravenous injection. Other injection routes, such as subcutaneous,intramuscular, or intraperitoneal, can also be used. More recently,alternative means for systemic administration of peptides have beendevised which include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if property formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thepatient's condition, and the judgment of the attending physician.Suitable dosage ranges, however, are in the range of 0.1-100 μg/kg ofsubject. Wide variations in the needed dosage, however, are to beexpected in view of the variety of peptides available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization as is well understood in the art.

The invention also relates to the therapeutic, prophylactic and researchuses of various techniques to block or modulate the expression of aP_(2U2) receptor by interfering with the transcription of translation ofa DNA or RNA molecule encoding the P_(2U2) receptor. This includes amethod to inhibit or regulate expression of P_(2U2) receptors in a cellcomprising providing to the cell an oligonucleotide molecule which isantisense to, or forms a triple helix with, P_(2U2) receptor-encodingDNA or with DNA regulating expression of P_(2U2) receptor-encoding DNA,in an amount sufficient to inhibit or regulate expression of the P_(2U2)receptors, thereby inhibiting or regulating their expression. Alsoincluded is a method to inhibit or regulate expression of P_(2U2)receptors in a subject, comprising administering to the subject anoligonucleotide molecule which is antisense to, or forms a triple helixwith, P_(2U2) receptor-encoding DNA or with DNA regulating expression ofP_(2U2) receptor-encoding DNA, in an amount sufficient to inhibit orregulate expression of the P_(2U2) receptors in the subject, therebyinhibiting or regulating their expression. The antisense molecule ortriple helix-forming molecule in the above methods is preferably a DNAor RNA oligonucleotide. These utilities are described in greater detailbelow.

The constitutive expression of antisense RNA in cells has been shown toinhibit the expression of about 20 different genes in mammals andplants, and the list continually grows (Hambor, et al., J. Exp. Med.168:1237-1245 (1988); Holt, et al., Proc. Natl. Acad. Sci. 83:4794-4798(1986); Izant, et al., Cell 36:1007-1015 (1984); Izant, et al., Science229:345-352 (1985) and De Benedetti, et al., Proc. Natl. Acad. Sci.84:658-662 (1987)). Possible mechanisms for the antisense effect are theblockage of translation or prevention of splicing, both of which havebeen observed in vitro. Interference with splicing allows the use ofintron sequences (Munroe, EMBO. J. 7:2523-2532 (1988) which should beless conserved and therefore result in greater specificity in inhibitingexpression of a protein of one species but not its homologue in anotherspecies.

Therapeutic gene regulation is accomplished using the “antisense”approach, in which the function of a target gene in a cell or organismis blocked, by transfection of DNA, preferably an oligonucleotide,encoding antisense RNA which acts specifically to inhibit expression ofthe particular target gene. The sequence of the antisense DNA isdesigned to result in a full or preferably partial antisense RNAtranscript which is substantially complementary to a segment of the geneor mRNA which it is intended to inhibit. The complementarity must besufficient so that the antisense RNA can hybridize to the target gene(or mRNA) and inhibit the target gene's function, regardless of whetherthe action is at the level of splicing, transcription or translation.The degree of inhibition, readily discernible by one of ordinary skillin the art without undue experimentation, must be sufficient to inhibit,or render the cell incapable of expressing, the target gene. One ofordinary skill in the art will recognize that the antisense RNA approachis but one of a number of known mechanisms which can be employed toblock specific gene expression.

By the term “antisense” is intended an RNA sequence, as well as a DNAsequence coding therefor, which is sufficiently complementary to aparticular mRNA molecule for which the antisense RNA is specific tocause molecular hybridization between the antisense RNA and the mRNAsuch that translation of the mRNA is inhibited. Such hybridization mustoccur under in vivo conditions, that is, inside the cell. The action ofthe antisense RNA results in specific inhibition of gene expression inthe cell. (See: Albers, et al., “Molecular Biology Of The Cell”, 2ndEd., Garland Publishing, Inc., New York, N.Y. (1989), in particular,pages 195-196).

The antisense RNA of the present invention may be hybridizable to any ofseveral portions of a target mRNA, including the coding sequence, a 3′or 5′ untranslated region, or other intronic sequences. A preferredantisense RNA is that complementary to the human P_(2U2) receptor mRNA.As is readily discernible by one of skill in the art, the minimal amountof homology required by the present invention is that sufficient toresult in hybridization to the specific target mRNA and inhibition ofits translation or function while not affecting function of other mRNAmolecules and the expression of other genes.

Antisense RNA is delivered to a cell by transformation or transfectionwith a vector into which has been placed DNA encoding the antisense RNAwith the appropriate regulatory sequences, including a promoter, toresult in expression of the antisense RNA in a host cell.

“Triple helix” or “triplex” approaches involve production of syntheticoligonucleotides which bind to the major groove of a duplex DNA to forma colinear triplex. Such triplex formation can regulate and inhibitcellular growth. See, for example: Hogan, et al., U.S. Pat. No.5,176,996; Cohen, et al., Sci. Amer., December 1994, p. 76-82; Helene,Anticancer Drug Design 6:569-584 (1991); Maher III, et al., AntisenseRes. Devel. 1:227-281 (Fall 1991); Crook, et al. eds., “AntisenseResearch and Applications”, CRC Press, 1993. It is based in part on thediscovery that a DNA oligonucleotide can bind by triplex formation to aduplex DNA target in a gene regulatory region, thereby repressingtranscription initiation (Cooney, et. al. Science 241:456 (1988)). Thepresent invention utilizes methods such as those of Hogan et al., supra(incorporated herein by reference in its entirety), to designingoligonucleotides which will bind tightly and specifically to a duplexDNA target comprising part of the P_(2U2) receptor-encoding DNA or aregulatory sequence thereof. Such triplex oligonucleotides can thereforebe used as a class of drug molecules to selectively manipulate theexpression of this gene.

Thus the present invention is directed to providing to a cell oradministering to a subject a synthetic oligonucleotide in sufficientquantity for cellular uptake and binding to a DNA duplex of the targetP_(2U2) receptor-coding DNA sequence or a regulatory sequence thereof,such that the oligonucleotide binds to the DNA duplex to form a colineartriplex. This method is used to inhibit expression of the receptor oncells in vitro or in vivo. Preferably the target sequence is positionedwithin the DNA domain adjacent to the RNA transcription origin. Thismethod can also be used to inhibit growth of cells which is dependent onexpression of this receptor. The method may also be used to alter therelative amounts or proportions of the P_(2U2) receptor expressed oncells or tissues by administering such a triplex-forming syntheticoligonucleotide.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 PCR (Polymerase Chain Reaction) Amplification of RelatedPurinergic Receptor cDNA with Degenerate Primers

DAMI cells (obtained from ATCC (# CRL9792)), were cultured in RPMI with10% fetal bovine serum, plus glutamine, penicillin/streptomycin andkanamycin, in 7% CO₂/93% air and mRNA was isolated by the guanidinethiocyanate method. Poly-A(+) mRNA was selected two times using oligo-dTcolumns (Stratagene). The twice-selected poly-A+mRNA was used togenerate first-strand cDNA by priming with either oligo-dT or randomprimers and AMV reverse transcriptase (Invitrogen) as a template forPCR. Primers were designed based on the sequence of transmembrane region3 (TM III, primer 3B) and transmembrane region 7 (TM VII, primer 7A2)from the mouse P_(2U) (Lustig, et al, Proc. Natl. Acad. Sci., USA90:5113-5117 (1993)) and the chicken P₂Y₁(Webb, et al, FEBS Letters324:219-225 (1993)) receptor. The nucleotide sequence of 3B (SEQ IDNO:9) was:

5′AT(CT)CT(GTC)TT(CT)CTGAC(CTA)TG(CT)AT(CT)(AT)(GC)IGT(GTC)CA 3′ and thesequence for 7A2 (SEQ ID NO:10) was:

3′GG(GAT)(TC)A(CGA)(GA)AIAT(GA)AA(AG)(GA)AICGICC5′

where G is guanine, C is cytosine, A is adenine, T is thymidine and I isinosine, and the “()” indicate positions of degeneracy such that thesequences were a mixture with the indicated substitutions at that givenposition. The following conditions were used for PCR using Taqpolymerase: 5 cycles of 93° C., 2 minutes; 60° C., 1.5 minutes; 72° C.,2.5 minutes; 5 cycles of 93° C., 2 minutes; 55° C., 1.5 minutes; 72° C.,2.5 minutes; 25 cycles of 93° C., 2 minutes; 50° C., 1.5 minutes; 72°C., 2.5 minutes, followed by a final extension of 72° C., 5 min. PCRproducts were purified over a size-selection column and ligated directlyinto the pCR2 TA cloning vector (Invitrogen) and the DNA was used totransform DH5α strain of E. coli. Colonies were selected and DNA wasprepared for restriction analysis and sequencing. Cycle sequencing wasperformed using Taq polymerase and dye-terminator mixes(Perkin-Elmer/ABI) and the results were analyzed on an ABI 373 automaticsequencer. Sequence results obtained with one clone, called 206.18,exhibited homology with published purinergic receptor sequences.

EXAMPLE 2 Isolation of Full-length Human cDNA Encoding P_(2U2)

Insert was isolated from the PCR clone of interest (206.18), purifiedfrom an agarose gel, radiolabeled with [α-³²P]dCTP(NEN) byrandom-priming (Stratagene), and used to screen a DAMI cDNA library inλgt22. The library was generated using twice-selected poly-A+mRNA (seeabove) and first strand cDNA synthesis was primed with an oligo-dTprimer and synthesized with Moloney murine leukemia virus (M-MLV)reverse transcriptase (Gibco/BRL). cDNA was directionally ligated intothe Sall/Notl sites of the λgt22 arms and packaged (Stratagene packagingextract) and amplified in the Y1090 (r-) strain. One million clones werescreened at a density of 40,000/plate under the following conditions:duplicate nitrocellulose filters (S&S) were hybridized overnight at 42°C. in a solution containing 50% deionized formamide, 5×SSC (sodiumchloride, sodium citrate), 0.1 mg/ml heat denatured salmon sperm DNA,0.1% sodium dodecylsulfate, 1×Denhardt's, 0.02M Tris, pH 7.5 and 1-2×106cpm/ml of radiolabeled probe. Filters were washed twice at roomtemperature for 10 minutes in 0.1% sodium dodecylsulfate, 2×SSC and thenat 55° C. for 30 minutes in 0.2×SSC, 0.1% sodium dodecylsulfate, thenexposed with an intensifying screen overnight at −70° C. with Kodak XARfilm. Positively hybridizing clones were plaque purified, λ DNA wasprepared and the cDNA inserts were excised and subcloned into thecommercially available pBluescript vector. The hybridizing and adjacentregions were sequenced on both strands as above on an ABI 373 automaticsequencer.

To isolate additional 5′ sequence for the P_(2U2) gene, a 5′ proximalfragment from the largest DAMI clone (D8) was used to screen a Clontechhuman kidney cDNA library (λgt10) under identical screening conditionsas were used for the DAMI cDNA library. DNA from plaque-purifiedpositively hybridizing clones from both libraries were analyzed byrestriction digest. Inserts from clones of interest were excised andsubcloned into the commercially available pBluescript vector andsequenced as above. The complete open reading frame as well as truncatedversions of the full-length cDNA were cloned into Xenopus oocyte ormammalian expression vectors for functional analysis. The DNA sequenceof the complete open reading frame for the longest cDNA isolated fromthe kidney cDNA library is shown in FIG. 1 (SEQ ID NO:1). As shown inFIG. 2, the deduced amino acid sequence of the P_(2U2) cDNA showsextensive homology with other known purinergic receptors (Parr, supra,and Henderson, supra).

EXAMPLE 3 Expression of P_(2U2) mRNA in Various Tissues and Cell Lines

Poly-(A)+RNA was isolated from a variety of cell lines as describedabove. Five μg of each sample was denatured, electrophoresed on a 1.2%formaldehyde agarose gel, and transferred to nylon membrane. Blots wereprobed with [α-³²P]dCTP(NEN) labeled insert, as described for thelibrary screenings, and hybridized at 42° C. overnight in the followingsolution: 5×SSPE, 10×Denhardt's, 50% formamide, 2% sodiumdodecylsulfate, 0.1 mg/ml heat denatured salmon sperm DNA. Blots werewashed twice at room temperature for 15 minutes in 0.05% SDS, 2×SSC andthen at 50° C. for 30 minutes in 0.1×SSC, 0.1% SDS and exposed at −70°C. to Kodak XAR film for 48-72 hours with an intensifying screen.Northern blots containing poly-A+RNA from human tissues were purchasedfrom Clontech and hybridized, washed and exposed as described above.Hybridization of RNA tissue blots with the labeled P_(2U2) DNA fragmentdemonstrated that a 4.4 kB mRNA is abundantly expressed in human kidney,but is negative for other tissues examined (heart, brain, placenta,lung, liver, skeletal muscle, pancreas, spleen, thymus, prostate,testis, ovary, small intestine, colon and peripheral blood leukocytes).These results distinguish this receptor from other reported purinergicreceptors since these other receptors are abundant in brain. A series ofmRNAs isolated from a variety of human hematopoietic and lymphocyticcell lines were used in a Northern analysis and a 4.4 kB message for thereceptor was demonstrated to be abundant in several cell lines oferythroleukemic (HEL, K562) and megakaryocytic (DAMI) origin, and notpresent in the monocytic cell line U937 or the T-cell derived Jurkatcell line.

EXAMPLE 4 Demonstration of the Function of the Receptor in Oocytes

The native human receptor was produced in oocytes by cloning the 500 bp5′ truncation of the full-length kidney cDNA clone into the mammalianexpression vector pcDNA3 (Invitrogen). Linearized DNA was used as atemplate for T7 polymerase (Ambion, Promega) for generation of capped invitro transcribed mRNA following the suppliers specifications. Adultfemale Xenopus laevis were anesthetized in [0.015 g/l] 3-aminobenzoicacid ethyl ester for 10 minutes and 1 or 2 ovarian lobes were removed,followed by immediate suturing of the incisions. Oocytes weredefolliculated at room temperature with collagenase (2 mg/ml) inCa⁺²-free medium (OR-2) for 1-2 hr. Oocytes were stored at 180° C. inND-96 (96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calciumchloride, 1 mM magnesium chloride, 5 mM HEPES(N[2-hydroxyethylpiperazine-N′[2-ethanesulfonic acid) withpenicillin/streptomycin and injected with 50 nl RNA (1-2 μg/μl) 18-24 hafter removal of the oocytes. Before recording, injected oocytes werestored at 18° C. for 2-3 days with daily media changes.

A two-electrode voltage clamp (Axon Axoclamp2B) was used to measureagonist-induced currents from individual oocytes. Electrodes were pulledto resisitances of 0.2-1 MΩ and filled with 3M KCl. Recordings were madeat room temperature in ND96 from oocytes clamped at −70 mV usingdifferent agonist concentrations. Water-injected oocytes were used as acontrol. FIG. 3 shows representative chloride currents obtained fromoocytes injected with cRNA for the P_(2U2) receptor and challenged witha variety of purinergic agonists (ADP, ATP, UTP, UDP).

All references cited and mentioned above, including patents, journalarticles and texts, are all incorporated by reference herein, whetherexpressly incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

14 1996 base pairs nucleic acid single linear cDNA CDS 625..1626 1ATAAAGTATG TTTAGCCCTC ATGTCACATG AACCTTTATG CATTGAAGAT TGTTTCCCTT 60GCCCCCCCAG GGGGTGGGGT TATTTTTCTA TCCTTGTTAA CTTCCCTATA TTATTATATA 120CACTTTGAGT TTTAGGGTAC ATGTGCACAA AGTGCAGGTT AGTTACATAT GTATACATGT 180GCCATGTTGG TGTGCTGCAC CCATTAACAC ATCATTTAGC ATGAGGTATA TCTCCTAATG 240TTATCCCTCC CCCCTCCCCC CACCCCACAA CAGTCCCCGG AGTGTGATAT TCCCCTTTCC 300TGTGTCCATG TGTTATTATT CCAATTCCCC ACCTATGAAG TGAAAATATG CAGGTGTTTG 360GATTTTTGTC CTTGGCAATA GTTTTGCTGA GAATGATGGT TTCCAGCTTC ATCCATGTCC 420CTACAAAGGA CATGAACTCA TCATTTTTTA TGACTGCATA GTATTCTATG GTGTATACAT 480GCCAACTTTT CTCCCCCCCC TTTTTAAGCT CCTTCTTTCA CTGGCTTTCA TGATCCCACC 540AATTCCTGCT TTTCCTTTTT TGTTTTTTTC TTCCAACAGA ATGGTTATGG TTTAACTCAG 600CAGAATTTGT TGAACAACTA CGAC ATG CTG GGG ATC ATG GCA TGG AAT GCA 651 MetLeu Gly Ile Met Ala Trp Asn Ala 1 5 ACT TGC AAA AAC TGG CTG GCA GCA GAGGCT GCC CTG GAA AAG TAC TAC 699 Thr Cys Lys Asn Trp Leu Ala Ala Glu AlaAla Leu Glu Lys Tyr Tyr 10 15 20 25 CTT TCC ATT TTT TAT GGG ATT GAG TTCGTT GTG GGA GTC CTT GGA AAT 747 Leu Ser Ile Phe Tyr Gly Ile Glu Phe ValVal Gly Val Leu Gly Asn 30 35 40 ACC ATT GTT GTT TAC GGC TAC ATC TTC TCTCTG AAG AAC TGG AAC AGC 795 Thr Ile Val Val Tyr Gly Tyr Ile Phe Ser LeuLys Asn Trp Asn Ser 45 50 55 AGT AAT ATT TAT CTC TTT AAC CTC TCT GTC TCTGAC TTA GCT TTT CTG 843 Ser Asn Ile Tyr Leu Phe Asn Leu Ser Val Ser AspLeu Ala Phe Leu 60 65 70 TGC ACC CTC CCC ATG CTG ATA AGG AGT TAT GCC AATGGA AAC TGG ATA 891 Cys Thr Leu Pro Met Leu Ile Arg Ser Tyr Ala Asn GlyAsn Trp Ile 75 80 85 TAT GGA GAC GTG CTC TGC ATA AGC AAC CGA TAT GTG CTTCAT GCC AAC 939 Tyr Gly Asp Val Leu Cys Ile Ser Asn Arg Tyr Val Leu HisAla Asn 90 95 100 105 CTC TAT ACC AGC ATT CTC TTT CTC ACT TTT ATC AGCATA GAT CGA TAC 987 Leu Tyr Thr Ser Ile Leu Phe Leu Thr Phe Ile Ser IleAsp Arg Tyr 110 115 120 TTG ATA ATT AAG TAT CCT TTC CGA GAA CAC CTT CTGCAA AAG AAA GAG 1035 Leu Ile Ile Lys Tyr Pro Phe Arg Glu His Leu Leu GlnLys Lys Glu 125 130 135 TTT GCT ATT TTA ATC TCC TTG GCC ATT TGG GTT TTAGTA ACC TTA GAG 1083 Phe Ala Ile Leu Ile Ser Leu Ala Ile Trp Val Leu ValThr Leu Glu 140 145 150 TTA CTA CCC ATA CTT CCC CTT ATA AAT CCT GTT ATAACT GAC AAT GGC 1131 Leu Leu Pro Ile Leu Pro Leu Ile Asn Pro Val Ile ThrAsp Asn Gly 155 160 165 ACC ACC TGT AAT GAT TTT GCA AGT TCT GGA GAC CCCAAC TAC AAC CTC 1179 Thr Thr Cys Asn Asp Phe Ala Ser Ser Gly Asp Pro AsnTyr Asn Leu 170 175 180 185 ATT TAC AGC ATG TGT CTA ACA CTG TTG GGG TTCCTT ATT CCT CTT TTT 1227 Ile Tyr Ser Met Cys Leu Thr Leu Leu Gly Phe LeuIle Pro Leu Phe 190 195 200 GTG ATG TGT TTC TTT TAT TAC AAG ATT GCT CTCTTC CTA AAG CAG AGG 1275 Val Met Cys Phe Phe Tyr Tyr Lys Ile Ala Leu PheLeu Lys Gln Arg 205 210 215 AAT AGG CAG GTT GCT ACT GCT CTG CCC CTT GAAAAG CCT CTC AAC TTG 1323 Asn Arg Gln Val Ala Thr Ala Leu Pro Leu Glu LysPro Leu Asn Leu 220 225 230 GTC ATC ATG GCA GTG GTA ATC TTC TCT GTG CTTTTT ACA CCC TAT CAC 1371 Val Ile Met Ala Val Val Ile Phe Ser Val Leu PheThr Pro Tyr His 235 240 245 GTC ATG CGG AAT GTG AGG ATC GCT TCA CGC CTGGGG AGT TGG AAG CAG 1419 Val Met Arg Asn Val Arg Ile Ala Ser Arg Leu GlySer Trp Lys Gln 250 255 260 265 TAT CAG TGC ACT CAG GTC GTC ATC AAC TCCTTT TAC ATT GTG ACA CGG 1467 Tyr Gln Cys Thr Gln Val Val Ile Asn Ser PheTyr Ile Val Thr Arg 270 275 280 GCT TTG GGC TTT CTG AAC AGT GTC ATC AACCCT GTC TTC TAT TTT CTT 1515 Ala Leu Gly Phe Leu Asn Ser Val Ile Asn ProVal Phe Tyr Phe Leu 285 290 295 TTG GGA GAT CAC TTC AGG GAC ATG CTG ATGAAT CAA CTG AGA CAC AAC 1563 Leu Gly Asp His Phe Arg Asp Met Leu Met AsnGln Leu Arg His Asn 300 305 310 TTC AAA TCC CTT ACA TCC TTT AGC AGA TGGGCT CAT GAA CTC CTA CTT 1611 Phe Lys Ser Leu Thr Ser Phe Ser Arg Trp AlaHis Glu Leu Leu Leu 315 320 325 TCA TTC AGA GAA AAG TGAGGGGCTTGTGAAACAGA TTGTTCTACA GATGAATCTG 1666 Ser Phe Arg Glu Lys 330 TAAGCCAGTTACAGTTTGCT TTAACTCATA GACATCAATC AGAGAGTGTC ACAGATTTAA 1726 CCTTGATCTAAAGACAAGTT GTACCCAGAG TATGTGAAAA GAATGGGACG ACAAGAATGT 1786 ACTGGTTTCTTCCTCTAAGA ATTGAAAGGA GTTGAACTGC CTTATGTTTG GGCATGTAAC 1846 TCCAAAATACTAGGTAGTAT AAGGCTTTCT CAATCAGTCC CCAAATGGAA GATATATAAA 1906 GCAACAAGTTGTCTGCATTT GATCACTGGT CAGATTGTAA AAAAAAAAAA AAAAAAGGGC 1966 GCCCGCCACCGCGGTGGAGC TCCAATCGCC 1996 334 amino acids amino acid linear protein 2Met Leu Gly Ile Met Ala Trp Asn Ala Thr Cys Lys Asn Trp Leu Ala 1 5 1015 Ala Glu Ala Ala Leu Glu Lys Tyr Tyr Leu Ser Ile Phe Tyr Gly Ile 20 2530 Glu Phe Val Val Gly Val Leu Gly Asn Thr Ile Val Val Tyr Gly Tyr 35 4045 Ile Phe Ser Leu Lys Asn Trp Asn Ser Ser Asn Ile Tyr Leu Phe Asn 50 5560 Leu Ser Val Ser Asp Leu Ala Phe Leu Cys Thr Leu Pro Met Leu Ile 65 7075 80 Arg Ser Tyr Ala Asn Gly Asn Trp Ile Tyr Gly Asp Val Leu Cys Ile 8590 95 Ser Asn Arg Tyr Val Leu His Ala Asn Leu Tyr Thr Ser Ile Leu Phe100 105 110 Leu Thr Phe Ile Ser Ile Asp Arg Tyr Leu Ile Ile Lys Tyr ProPhe 115 120 125 Arg Glu His Leu Leu Gln Lys Lys Glu Phe Ala Ile Leu IleSer Leu 130 135 140 Ala Ile Trp Val Leu Val Thr Leu Glu Leu Leu Pro IleLeu Pro Leu 145 150 155 160 Ile Asn Pro Val Ile Thr Asp Asn Gly Thr ThrCys Asn Asp Phe Ala 165 170 175 Ser Ser Gly Asp Pro Asn Tyr Asn Leu IleTyr Ser Met Cys Leu Thr 180 185 190 Leu Leu Gly Phe Leu Ile Pro Leu PheVal Met Cys Phe Phe Tyr Tyr 195 200 205 Lys Ile Ala Leu Phe Leu Lys GlnArg Asn Arg Gln Val Ala Thr Ala 210 215 220 Leu Pro Leu Glu Lys Pro LeuAsn Leu Val Ile Met Ala Val Val Ile 225 230 235 240 Phe Ser Val Leu PheThr Pro Tyr His Val Met Arg Asn Val Arg Ile 245 250 255 Ala Ser Arg LeuGly Ser Trp Lys Gln Tyr Gln Cys Thr Gln Val Val 260 265 270 Ile Asn SerPhe Tyr Ile Val Thr Arg Ala Leu Gly Phe Leu Asn Ser 275 280 285 Val IleAsn Pro Val Phe Tyr Phe Leu Leu Gly Asp His Phe Arg Asp 290 295 300 MetLeu Met Asn Gln Leu Arg His Asn Phe Lys Ser Leu Thr Ser Phe 305 310 315320 Ser Arg Trp Ala His Glu Leu Leu Leu Ser Phe Arg Glu Lys 325 330 375amino acids amino acid <Unknown> linear protein 3 Met Ala Ala Asp LeuGly Pro Trp Asn Asp Thr Ile Asn Gly Thr Trp 1 5 10 15 Asp Gly Asp GluLeu Gly Tyr Arg Cys Arg Phe Asn Glu Asp Phe Lys 20 25 30 Tyr Val Leu LeuPro Val Ser Tyr Gly Val Val Cys Val Leu Gly Leu 35 40 45 Cys Leu Asn AlaVal Gly Leu Tyr Ile Phe Leu Cys Arg Leu Lys Thr 50 55 60 Trp Asn Ala SerThr Thr Tyr Met Phe His Leu Ala Val Ser Asp Ala 65 70 75 80 Leu Tyr AlaAla Ser Leu Pro Leu Leu Val Tyr Tyr Tyr Ala Arg Gly 85 90 95 Asp His TrpPro Phe Ser Thr Val Leu Cys Lys Leu Val Arg Phe Leu 100 105 110 Phe TyrThr Asn Leu Tyr Cys Ser Ile Leu Phe Leu Thr Cys Ile Ser 115 120 125 ValHis Arg Cys Leu Gly Val Leu Arg Pro Leu Arg Ser Leu Arg Trp 130 135 140Gly Arg Ala Arg Tyr Ala Arg Arg Val Ala Gly Ala Val Trp Val Leu 145 150155 160 Val Leu Ala Cys Gln Ala Pro Val Leu Tyr Phe Val Thr Thr Ser Ala165 170 175 Arg Gly Pro Leu Thr Cys His Asp Thr Ser Ala Pro Glu Leu PheSer 180 185 190 Arg Phe Val Ala Tyr Ser Ser Val Met Leu Gly Leu Leu PheAla Val 195 200 205 Pro Phe Ala Val Ile Leu Val Cys Tyr Val Leu Met AlaArg Arg Leu 210 215 220 Leu Lys Pro Ala Tyr Gly Thr Ser Gly Gly Leu ProArg Ala Lys Arg 225 230 235 240 Lys Ser Val Arg Thr Ile Ala Val Val LeuAla Val Phe Ala Leu Cys 245 250 255 Phe Leu Pro Phe His Val Thr Arg ThrLeu Tyr Tyr Ser Phe Arg Ser 260 265 270 Leu Asp Leu Ser Cys His Thr LeuAsn Ala Ile Asn Met Ala Tyr Lys 275 280 285 Val Thr Arg Leu Ala Ser AlaAsn Ser Cys Leu Asp Pro Val Leu Tyr 290 295 300 Phe Leu Ala Gly Gln ArgLeu Val Arg Phe Ala Arg Asp Ala Lys Pro 305 310 315 320 Pro Thr Gly ProSer Pro Ala Thr Pro Ala Arg Arg Thr Leu Gly Leu 325 330 335 Arg Arg SerAsp Arg Thr Asp Met Gln Arg Ile Gly Asp Val Leu Gly 340 345 350 Ser SerGlu Asp Ser Arg Arg Thr Glu Ser Thr Pro Ala Gly Ser Glu 355 360 365 AsnThr Lys Asp Ile Arg Leu 370 375 373 amino acids amino acid <Unknown>linear protein 4 Met Thr Glu Val Leu Trp Pro Ala Val Pro Asn Gly Thr AspThr Ala 1 5 10 15 Phe Leu Ala Asp Pro Gly Ser Pro Trp Gly Asn Ser ThrVal Thr Ser 20 25 30 Thr Ala Ala Val Ala Ser Pro Phe Lys Cys Ala Leu ThrLys Thr Gly 35 40 45 Phe Gln Phe Tyr Tyr Leu Pro Ala Val Tyr Ile Leu ValPhe Ile Ile 50 55 60 Gly Phe Leu Gly Asn Ser Val Ala Ile Trp Met Phe ValPhe His Met 65 70 75 80 Lys Pro Trp Ser Gly Ile Ser Val Tyr Met Phe AsnLeu Ala Leu Ala 85 90 95 Asp Phe Leu Tyr Val Leu Thr Leu Pro Ala Leu IlePhe Tyr Tyr Phe 100 105 110 Asn Lys Thr Asp Trp Ile Phe Gly Asp Ala MetCys Lys Leu Gln Arg 115 120 125 Phe Ile Phe His Val Asn Leu Tyr Gly SerIle Leu Phe Leu Thr Cys 130 135 140 Ile Ser Ala His Arg Tyr Ser Gly ValVal Tyr Pro Leu Lys Ser Leu 145 150 155 160 Gly Arg Leu Lys Lys Lys AsnAla Val Tyr Ile Ser Val Leu Val Trp 165 170 175 Leu Ile Val Val Val GlyIle Ser Pro Ile Leu Phe Tyr Ser Gly Thr 180 185 190 Gly Ile Arg Lys AsnLys Thr Ile Thr Cys Tyr Asp Thr Thr Ser Asp 195 200 205 Glu Tyr Leu ArgSer Tyr Phe Ile Tyr Ser Met Cys Thr Thr Val Ala 210 215 220 Met Phe CysVal Pro Leu Val Leu Ile Leu Gly Cys Tyr Gly Leu Ile 225 230 235 240 ValArg Ala Leu Ile Tyr Lys Asp Leu Asp Asn Ser Pro Leu Arg Arg 245 250 255Lys Ser Ile Tyr Leu Val Ile Ile Val Leu Thr Val Phe Ala Val Ser 260 265270 Tyr Ile Pro Phe His Val Met Lys Thr Met Asn Leu Arg Ala Arg Leu 275280 285 Asp Phe Gln Thr Pro Glu Met Cys Ala Phe Asn Asp Arg Val Tyr Ala290 295 300 Thr Tyr Gln Val Thr Arg Gly Leu Ala Ser Leu Asn Ser Cys ValAsp 305 310 315 320 Pro Ile Leu Tyr Phe Leu Ala Gly Asp Thr Phe Arg ArgArg Leu Ser 325 330 335 Arg Ala Thr Arg Lys Ala Ser Arg Arg Ser Glu AlaAsn Leu Gln Ser 340 345 350 Lys Ser Glu Asp Met Thr Leu Asn Ile Leu SerGlu Phe Lys Gln Asn 355 360 365 Gly Asp Thr Ser Leu 370 23 amino acidsamino acid <Unknown> linear peptide 5 Met Leu Gly Ile Met Ala Trp AsnAla Thr Cys Lys Asn Trp Leu Ala 1 5 10 15 Ala Glu Ala Ala Leu Glu Lys 2017 amino acids amino acid <Unknown> linear peptide 6 Tyr Ala Asn Gly AsnTrp Ile Tyr Gly Asp Val Leu Cys Ile Ser Asn 1 5 10 15 Arg 22 amino acidsamino acid <Unknown> linear peptide 7 Asn Pro Val Ile Thr Asp Asn GlyThr Thr Cys Asn Asp Phe Ala Ser 1 5 10 15 Ser Gly Asp Pro Asn Tyr 20 20amino acids amino acid <Unknown> linear peptide 8 Ala Ser Arg Leu GlySer Trp Lys Gln Tyr Gln Cys Thr Gln Val Val 1 5 10 15 Ile Asn Ser Phe 2029 base pairs nucleic acid single linear other nucleic acid /desc =“primer” 9 ATYCTBTTYC TGACHTGYAT YWSNGTBCA 29 23 base pairs nucleic acidsingle linear other nucleic acid /desc = “primer” 10 CCNGCNARRAARTANARVAY DGG 23 11 amino acids amino acid <Unknown> linear peptide 11Phe Ser Leu Lys Asn Trp Asn Ser Ser Asn Ile 1 5 10 22 amino acids aminoacid <Unknown> linear peptide 12 Arg Tyr Leu Ile Ile Lys Tyr Pro Phe ArgGlu His Leu Leu Gln Lys 1 5 10 15 Lys Glu Phe Ala Ile Leu 20 26 aminoacids amino acid <Unknown> linear peptide 13 Tyr Lys Ile Ala Leu Phe LeuLys Gln Arg Asn Arg Gln Val Ala Thr 1 5 10 15 Ala Leu Pro Leu Glu LysPro Leu Asn Leu 20 25 34 amino acids amino acid <Unknown> linear peptide14 His Phe Arg Asp Met Leu Met Asn Gln Leu Arg His Asn Phe Lys Ser 1 510 15 Leu Thr Ser Phe Ser Arg Trp Ala His Glu Leu Leu Leu Ser Phe Arg 2025 30 Glu Lys

We claim:
 1. An isolated antibody or antigen binding fragment thatspecifically binds to a P_(2U2) purinergic receptor protein of SEQ IDNO: 2 which is activated by four agonists in the following order ofspecificity: UTP>UDP>ADP>ATP.
 2. An isolated antibody of claim 1,wherein the antibody is a monoclonal antibody.
 3. An isolated antibodyof claim 1, wherein the P_(2U2) purinergic receptor protein is a humanP_(2U2) receptor protein.
 4. An isolated antigen binding fragment ofclaim 1, wherein said fragment is selected from the group consisting ofFab, Fv, F(ab′)₂, and Fab′.
 5. A pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient and an antibody orantigen binding fragment of claim
 1. 6. An isolated antibody or antigenbinding fragment of claim 1, wherein the antibody or antigen bindingfragment specifically binds to an external loop segment of the receptorprotein.
 7. An isolated antibody of claim 1, wherein the antibody is achimeric antibody.
 8. An isolated antibody of claim 7, wherein theantibody is humanized.